Purpose The purpose of this study was to investigate 13C hyperpolarization of α-ketobutyrate (αKB), an endogenous molecular analog of pyruvate, and its in vivo enzymatic conversion via lactate dehydrogenase (LDH) using localized MR spectroscopy. Methods Hyperpolarized (HP) 13C MR experiments were conducted using [13C]αKB with rats in vivo and with isolated LDH enzyme in vitro, along with comparative experiments using [13C]pyruvate. Based on differences in the kinetics of its reaction with individual LDH isoforms, HP [13C]αKB was investigated as a novel MR probe, with added specificity for activity of LDHB-expressed H ("heart"-type) subunits of LDH (e.g., constituents of LDH-1 isoform). Results Comparable T1 and polarization values to pyruvate were attained (T1 = 52 s at 3 tesla [T], polarization = 10%, at C1). MR experiments showed rapid enzymatic conversion with substantially increased specificity. Formation of product HP [13C]α-hydroxybutyrate (αHB) from αKB in vivo was increased 2.7-fold in cardiac slabs relative to liver and kidney slabs. In vitro studies resulted in 5.0-fold higher product production from αKB with bovine heart LDH-1, as compared with pyruvate. Conclusions HP [13C]αKB may be a useful MR probe of cardiac metabolism and other applications where the role of H subunits of LDH is significant (e.g., renal cortex and brain).